RAGE Mediated Mechanisms of Islet Amyloid Polypeptide Cytotoxicity in Type 2 Diab

RAGE介导的2型糖尿病中胰岛淀粉样多肽细胞毒性的机制

• 批准号: 8004404
• 负责人: Andisheh Abedini
• 金额: $ 5.22万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-23 至 2012-07-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004404
• 关键词: Advanced Glycosylation End Products; Affect; Alzheimer' s Disease; Amyloid; Amyloidosis; Binding; Biochemical; Biological; Biological Assay; Biophysics; Brain; Cell Death; Cell physiology; Cell surface; Cells; Cessation of life; Chronic; Data; Diabetes Mellitus; Disease; Endocrine; Extracellular Domain; Fluorescence; Goals; Graft Survival; Homeostasis; Hormones; Human; In Vitro; Individual; Islets of Langerhans; Islets of Langerhans Transplantation; Kinetics; Learning; Ligands; Malignant Neoplasms; Mediating; Methods; Molecular; Molecular Conformation; Nature; Neurologic Dysfunctions; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Oxidative Stress; Pancreas; Parkinson Disease; Pathogenesis; Peptides; Physiological; Play; Prevention; Principal Investigator; Process; Receptor Activation; Recombinants; Recruitment Activity; Role; Site; Staging; System; Testing; Thioflavin T; Time; Toxic effect; Translating; Work; amyloid formation; cytotoxicity; follow-up; human disease; inhibitor/antagonist; insight; interdisciplinary approach; islet; islet amyloid polypeptide; light scattering; monomer; mutant; neurotoxic; polypeptide; public health relevance; receptor; receptor binding; treatment strategy

DESCRIPTION (provided by applicant): Amyloid formation plays an important role in a broad range of human diseases, including Alzheimer's disease (AD), Parkinson's disease, and type 2 diabetes (T2D). Islet amyloid polypeptide (known as amylin or IAPP) is an endocrine hormone responsible for pancreatic amyloid formation in T2D. The process of amyloid formation by human amylin (h-amylin) promotes b-cell death thereby contributing to the loss of 2-cell mass associated with advanced stages of T2D. There is increasing evidence that islet amyloid formation is an important complicating factor in islet cell transplantation, and we have recently shown that prevention of islet amyloid can contribute significantly to graft survival. My goals are to define the nature of the toxic species produced during amyloid formation by h-amylin and to identify the mechanism of islet amyloid induced toxicity in T2D. Biochemical and physiological studies have shown that b-sheet containing amyloid precursors can bind and activate RAGE (receptor for advanced glycation end-products). RAGE is a multi-ligand receptor involved in a diverse range of chronic human disorders including cancers, AD and diabetes. RAGE is a potential cell-surface acceptor site for the neurotoxic amyloid-b (Ab) peptides in the brain of individuals with AD, and plays an important role in the pathogenesis of neurological dysfunction and death. Ab amyloid accumulation in the brain increases with RAGE expression, and activation of RAGE is associated with sustained cellular oxidative stress which adversely affects cellular function and organic homeostasis. Given the similar polypeptide sequences and aggregation kinetics of h-amylin and Ab, I hypothesize that RAGE may also be a pathological receptor for h-amylin; and that activation of RAGE by h-amylin binding is a mechanism of islet amyloid toxicity in T2D. To test this hypothesis, I will apply a multidisciplinary approach which utilizes biological, biophysical and biochemical methods. We have recruited the assistance of a world leader in amylin biophysics to assist us and help guide my studies. My preliminary data show that h-amylin binds RAGE in vitro. I will follow up these results by determining which form of h-amylin (monomer, oligomer and/or amyloid) binds to RAGE and/or has the strongest interaction with RAGE. As part of this aim, I will also identify which extracellular domain(s) of RAGE interact(s) with h-amylin using recombinant soluble RAGE (sRAGE) mutants with one or more domain deletions. Independent of the first aim, I will determine whether h-amylin cytotoxicity is RAGE-mediated. To do this I will first identify the form of h-amylin (monomer, oligomer and/or amyloid) is responsible for cytotoxicity during amyloid formation by using thioflavin-T fluorescence assays, far-UV CD, light scattering and TEM in conjunction with time-dependent assays of cytotoxicity. I will determine the role of RAGE in h-amylin mediated cytotoxicity. These studies will make use of inhibitors of h-amylin toxicity and amyloid formation, as well as mutants of h-amylin which oligomerize but are not toxic. Molecules which trap specific toxic conformations populated by h-amylin during amyloid formation will also be used. The ability of these species to bind the soluble extracellular domain of recombinant human sRAGE will be tested and their effects on cultured RAGE +/+ and RAGE -/- primary cells will be analyzed. This work will provide a molecular level understanding of a receptor-mediated mechanism of islet amyloid toxicity in T2D. The studies will provide a nonbiased strategy for investigating amyloid-RAGE interactions that can be applied to other systems. The lessons learned will not only provide insight into potential strategies for the treatment and prevention of T2D, but may also be translated to better controlling pathological amyloidosis associated with other diseases. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: The goal of this project is to determine whether RAGE plays a role in the pathological amyloid formation and toxicity of human amylin, which is associated with pancreatic islet amyloid and type 2 diabetes. The lessons learned from the proposed studies will provide a nonbiased strategy for investigating amyloid-RAGE interactions that can be applied to other systems. The outcomes of the proposed studies will provide insight into potential strategies for the treatment and prevention of T2D in particular, but may also be translated to better controlling pathological amyloidosis associated with other diseases. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

描述（由申请人提供）：淀粉样蛋白形成在广泛的人类疾病中起重要作用，包括阿尔茨海默病（AD），帕金森病和2型糖尿病（T2D）。胰岛淀粉样蛋白多肽（胰淀素或IAPP）是一种内分泌激素，负责胰岛淀粉样蛋白的形成。人胰淀素（h-胰淀素）形成淀粉样蛋白的过程促进b细胞死亡，从而导致与T2D晚期相关的2细胞质量损失。越来越多的证据表明，胰岛淀粉样蛋白的形成是胰岛细胞移植的一个重要的复杂因素，我们最近表明，预防胰岛淀粉样蛋白可以显著促进移植物的存活。我的目标是确定h-胰淀素在淀粉样蛋白形成过程中产生的有毒物质的性质，并确定胰岛淀粉样蛋白诱导T2D毒性的机制。生化和生理学研究表明，含有淀粉样蛋白前体的b-sheet可以结合并激活RAGE（晚期糖基化终产物受体）。RAGE是一种多配体受体，参与多种慢性人类疾病，包括癌症、AD和糖尿病。RAGE是AD患者大脑中神经毒性淀粉样蛋白b （Ab）肽的潜在细胞表面受体位点，在神经功能障碍和死亡的发病机制中起重要作用。大脑中Ab淀粉样蛋白的积累随着RAGE的表达而增加，RAGE的激活与持续的细胞氧化应激有关，这对细胞功能和有机稳态产生不利影响。鉴于h-amylin和Ab相似的多肽序列和聚集动力学，我假设RAGE也可能是h-amylin的病理受体；通过h-胰淀素结合激活RAGE是T2D胰岛淀粉样蛋白毒性的机制之一。为了验证这一假设，我将采用多学科方法，利用生物学、生物物理学和生物化学方法。我们已经聘请了一位世界顶级的胰淀素生物物理学家来协助我们并指导我的研究。我的初步数据显示h-胰淀素能在体外与RAGE结合。我将通过确定哪种形式的h-胰淀素（单体、低聚物和/或淀粉样蛋白）与RAGE结合和/或与RAGE相互作用最强来跟踪这些结果。作为这一目标的一部分，我还将使用重组可溶性RAGE （sRAGE）突变体确定RAGE的哪些细胞外结构域与h-amylin相互作用，这些结构域存在一个或多个缺失。独立于第一个目标，我将确定h-胰淀素细胞毒性是否由rage介导。为此，我将首先通过使用硫黄- t荧光测定、远紫外CD、光散射和透射电镜结合时间依赖性细胞毒性测定，确定在淀粉样蛋白形成过程中负责细胞毒性的h-胰淀素（单体、低聚物和/或淀粉样蛋白）的形式。我将确定RAGE在h-胰淀素介导的细胞毒性中的作用。这些研究将利用抑制h-amylin毒性和淀粉样蛋白形成的抑制剂，以及寡聚但无毒的h-amylin突变体。在淀粉样蛋白形成过程中捕获由h-amylin填充的特定有毒构象的分子也将被使用。这些物种结合重组人sRAGE的可溶性胞外结构域的能力将被测试，并分析它们对培养的RAGE +/+和RAGE -/-原代细胞的影响。这项工作将在分子水平上了解受体介导的胰岛淀粉样蛋白毒性机制。这些研究将为研究淀粉样蛋白- rage相互作用提供一种无偏见的策略，这种策略可以应用于其他系统。所获得的经验教训不仅将为T2D的治疗和预防提供潜在的策略，而且还可能转化为更好地控制与其他疾病相关的病理性淀粉样变性。小灵通398/2590 （Rev. 06/09）页延续格式页